Peptide for Determining Actin Structures in Living Cells

ABSTRACT

The present invention relates to novel peptides capable of binding to action. The peptides are useful in methods for detecting actin in vitro or in living cells.

The present invention relates to novel peptides capable of binding toactin. The peptides are useful in methods for detecting actin in vitroor in living cells.

Actin is involved in many cellular processes such as morphogenesis,intracellular transport, cell division, muscle contraction and cellmigration. The actin cytoskeleton is also altered in disease processessuch as in invading tumour cells, myopathies or polycystic kidneydisease⁽¹⁻³⁾. In studying the various mentioned processes and conditionsa reliable staining of the actin cytoskeleton is essential. In mostcases staining of fixed cells with the F-actin binding compoundphalloidin coupled to fluorescent dyes is used. However, some cells suchas the plant pathogenic fungus Ustilago maydis, cannot be stained byphalloidin⁽⁴⁾. In addition it is often desirable to image actin inliving cells. The dynamics of actin filaments are much more sensitivereadouts for cytoskeletal organization and processes such as cellpolarization or cell migration can only be properly studied throughanalysis of cytoskeletal dynamics.

To study actin dynamics, researchers have relied either on the injectionof fluorescently labelled actin or small amounts of phalloidin⁽⁵⁻⁶⁾ oron the use of GFP fusion proteins. In the former case application islimited to large cells that can be injected, requires specializedequipment, relatively expensive probes and quantitative analysis iscomplicated by difficult control of the fluorescent actin concentration.Speckle analysis has provided a powerful tool for the detailed study ofactin polymerization dynamics with only trace amounts of labelledmolecules. Various GFP fusion proteins have been used to visualize actinin living cells. Most often actin itself has been fused to GFP but alldocumented actin-GFP fusion proteins exhibit reduced functionality andthey can only be used in combination with non-tagged actin present⁽⁷⁾.Even in cases where cells are not visibly affected by actin-GFPexpression it is not certain whether actin dynamics is partiallyaffected. Therefore, while actin-GFP can be used in several cell typesto monitor actin distribution it is to be used with great care ifstudying actin dynamics. Actin-GFP is also limited in its application asit exhibits a strong background staining from labelled actin monomersand therefore requires low expression levels.

Alternatively fusion of GFP to several actin binding domains have beenused, notably from moesin in Drosophila ⁽⁸⁾, LimE in Dictyostelium⁽⁹⁾,Abp120 in Dictyostelium and mammalian cells^((10,11)) and utrophin inXenopus ⁽¹²⁾. In plants fusions to the actin binding domains of mousetalin⁽¹³⁾ or fimbrin⁽¹⁴⁾ have been used but each seems to stain only asubset of actin structures and can lead to artificial bundling of actinif expressed at high levels^((15,16)). In general the used fusionproteins are still quite large and are restricted to cells that can betransfected or injected.

The present invention describes the identification and application ofshort novel peptides, which specifically stain F-actin structures in awide range of cell types when coupled to labelling groups such asfluorescent proteins or chemical labelling groups. Preferably, thesenovel peptides bind efficiently to actin and do not interfere with actindynamics in vitro and in vivo. The novel peptides were validated byvisualizing and quantifying actin dynamics in several sensitivebiological processes such as neuronal growth cone formation or dendriticcell migration. The novel peptides of the present invention may beeasily obtained by chemical synthesis or recombinant methods and allowvisualization of actin dynamics in non-transfectable cells, such asprimary neutrophils.

The novel peptide probes are ideally suited as a marker for F-actin inliving as well as fixed cells. Preferably, the probe is a 17 amino acid(aa) fragment of the yeast actin binding protein Abp140 that faithfullystains F-actin without interfering with actin polymer dynamics.

A first aspect of the present invention refers to a peptide having theamino acid sequence (I)

(M)_(n)GVADLIKKFEwherein n is 0 or 1, or a variant thereof.

A further aspect of the present invention refers to a peptide having theamino acid sequence (II)

(M)_(n)GVADLIKKFESISKEEwherein n is 0 or 1, or a variant thereof.

Amino acid sequences (I) and (II) are derived from the N-terminalportion of the actin binding protein Abp140 from the yeast S.cerevisiae. In addition to these sequences, the present invention refersalso to variants thereof, e.g. to variants derived from other fungalspecies, e.g. as disclosed FIG. 1 e of the present application. Inpreferred variants of the peptides (I) and (II), amino acid residues2-10 are selected such that an α-helical structure is formed. Further,the invention encompasses peptides with one, two, three or even moreamino acid substitutions within amino acid residues 2-11 of peptides (I)and (II) and optionally one, two, three, four or even more amino acidsubstitutions within amino acid residues 12-17 of peptide (II). Forexample, in peptide (I) the amino acid residue G at position 2 may bereplaced by S and/or the amino acid residue K at position 8 may bereplaced by Q or R. In peptide (II) additional amino acid substitutionsmay occur e.g. at positions 12 to 17, e.g. a substitution of S atposition 12 by K, Q, D and/or T, a substitution of I at position 13 by For Y, a substitution of S at position 14 by A or T, a substitution of Kat position 15 by N, H, or Q, a substitution of E at position 16 by K,V, S or D, and/or a substitution of E at position 17 by K, D, S, Gand/or P.

The peptides of the present invention are preferably capable ofefficiently binding to actin, particularly to F-actin. The dissociationconstant K_(d) is usually ≦50 μM, preferably ≦10 μM and more preferably≦5 μM.

The peptides of the invention may be linear or cyclic peptides.Optionally the peptides may comprise modified or non-genetically encodedamino acid residues. The peptides preferably have a length of up to 120,preferably up to 50, and more preferably up to 20 amino acid residues.More preferably, the peptides are selected from the N-terminal portionof fungal Abp140 polypeptides, e.g. from S. cerevisiae, or actin-bindingfragments thereof.

The peptides of the invention may be monomeric or multimeric.Multimerisation of the peptides may comprise the use of linkers. Thelinkers may be naturally occurring or synthetically produced peptidesequences having a length of e.g. 1 to 15, preferably 1 to 10 aminoacids. Alternatively, the linkers may be non-peptidic moieties, e.g.synthetic amino acids, chemical linkers, oligo- or poly(alkylene)oxidemoieties.

The peptides are preferably coupled to heterologous molecules, e.g.labelling groups, which allow detection of the peptides. In oneembodiment, the heterologous molecule is a peptide or polypeptide, e.g.a labelling peptide such as the FLAG epitope or a labelling polypeptidesuch as GFP or any variant thereof. In another embodiment, theheterologous molecule may be a non-peptidic molecule, e.g. a chemicallabelling group, particularly a fluorescent chemical labelling groupsuch as fluoresceine, rhodamine or another fluorescent labelling group.The heterologous molecule may be coupled to the N- and/or to theC-terminus of the peptides (I) or (II). For example, fluorescentpolypeptides such as GFP or mRFPruby may be genetically fused to theC-terminus of the peptide without affecting actin binding. Further,fluoresceine isothiocyanate (FITC) may be coupled to the N-terminus ofthe peptides also without affecting binding to actin. Furthermore,peptidic linker sequences such as the sequence C-G may be added to theC-terminus for functional coupling of heterologous molecules via theSH-group of cysteine.

The peptides of the invention may be synthesized by chemical methods,e.g. using a solid phase peptide synthesizer or—particularly in the caseof peptides fused to heterologous peptides or polypeptides—byrecombinant methods in suitable host cells.

Thus, a further aspect of the present invention refers to a nucleic acidmolecule encoding a peptide as described above, particularly a peptidefused to a heterologous peptide or polypeptide, particularly to afluorescent polypeptide. For example, the nucleic acid molecule may be asingle- or double-stranded DNA or RNA molecule. Preferably, the nucleicacid molecule is operatively linked to an expression control sequence,i.e. a sequence which is sufficient for effecting expression in asuitable host cell, e.g. a prokaryotic or eukaryotic host cell.Preferably, the expression control sequence is capable of providingexpression in a vertebrate host cell, particularly in a mammalian hostcell.

Still a further aspect of the present invention refers to recombinantcells transfected or transformed with a nucleic acid molecule asdescribed above. The recombinant cell may be a prokaryotic cell, e.g. anE. coli cell, or a eukaryotic cell, e.g. a fungal cell, an animal cell,particularly a vertebrate cell such as an insect, fish, bird ormammalian cell or a plant cell. Transfection or transformation of therecombinant cell may be carried out by standard methods known in theart, e.g. calcium phosphate precipitation or electroporation.

Still a further aspect of the present invention is a non-humantransgenic organism transfected or transformed with the nucleic acidmolecule as described above. The transgenic organism may be a fungus, ananimal, e.g. a nematode like C. elegans, an insect such as Drosophila, afish such as Danio rero, or a mammal such as a rodent, e.g. a mouse or arat. On the other hand, the organism may be a plant such a Arabidopsisthaliana.

The peptide, nucleic acid molecule, recombinant cell or non-humantransgenic organism as described above are useful for the detection ofactin, preferably for the detection of actin within a cell, particularlywithin a living cell. By means of the present invention,actin-associated processes such as cell migration, cell division and/orcell differentiation may be detected. Other relevant applications areresearch on cell polarity, neuronal development, muscle development,cell-matrix interaction, cancerogenesis, immune reactions, e.g. ofneutrophils, T-cells or dendritic cells etc.

Further, the present invention has also applications in pharmaceuticalresearch. As the actin cytoskeleton is a key component for nearly alltypes of cellular morphogenesis, actin may be used as marker or targetin cell-based screening methods and therapeutic approaches. Thus, thepresent invention is suitable for application in cell-based screens fordrugs, e.g. cancer drugs, or in the study of actin-based diseases suchas Myopathies or polycystic kidney disease.

Furthermore, the peptides of the present invention may be used for themodulation of actin-associated processes in therapeutic applications. Inthis embodiment, the peptides may serve as targeting molecules for theactin cytoskeleton. The peptides may be coupled to effector groups suchas oxygen radical producers or caged compounds which can be activated ina regulated way. Further, cellular signalling molecules or cellularregulators or cytoskeleton modulators may be used as effector groups, toredirect cellular functions and/or cellular structure. For example,actin nucleators, organelle membrane proteins, GTPase regulators such asactivators of Rho-type GTPases may be linked to the peptides of theinvention and targeted to actin, thereby increasing the effects onactin-dependent processes.

Further, multimeric peptides, e.g. dimeric or trimeric peptides, may beused to modulate cellular actin structures and thus cellular processes.

Furthermore, the above-described peptides may be used in drug screening,particularly when coupled to a labeling group. Particularly preferred isthe use in high throughput drug screening assays as a readout system todetect phenotypic responses of a cell to a screening compound, e.g.alterations in the cellular actin structure.

Further, the present invention shall be explained in more detail by thefollowing Figures and Examples.

FIGURE LEGEND

FIG. 1 Identification of Lifeact. a) TIRF image of Abp140-GFPdistribution in an unpolarized yeast cell. Scale bar: 5 μm. b) Fastblinking of Abp140-GFP signals on actin structures. Time in ms, scalebar: 2 μm. c) distribution of the minimal Lifeact-G in a yeast cell.Scale bar: 5 μm. d) Schematic organization of Abp140 and illustration oftested localization constructs. Numbers indicate amino acid positions.Localization scores: ++comparable to full-length protein, − notlocalized, +/− localizes mostly to actin patches. e) ClustalW alignmentof Lifeact with homologous sequences from related fungi.

FIG. 2 Biochemistry of Lifeact. a) Measurement of F-Lifeact binding torabbit muscle F-actin. Shown is the relative fluorescence of peptideco-sedimented with various concentrations of F-actin. b) Measurement ofF-Lifeact binding to G-actin. Binding was determined by measuringrelative changes in pyrene-G-actin fluorescence in the presence ofvarying amounts of F-Lifeact. c) Actin polymerization assay.Polymerization of pyrene-actin was followed in the presence of indicatedconcentrations of F-Lifeact. d) Actin depolymerization assay.Depolymerization of pyrene-F-actin was followed after dilution below thecritical concentration in the presence of indicated concentrations ofF-Lifeact. e) Circular Dichroism (CD) measurements on F-Lifeact upontitration with 0-50% TFE. Inset: CD on F-Lifeact and Lifeact withoutTFE. f) Short and medium range NOE connectivities involving the NH andC^(α)H protons (21,29). Blue bars represent measurements on F-Lifeact atpH 7.1, red bars represent measurements on Lifeact at pH 3.0 in thepresence of 15% (v/v) HFP-d₂.

FIG. 3 Lifeact-GFP: visualization and functional characterization invivo. a-j) Lifeact G was transiently expressed in mouse embryonicfibroblasts (a, j), primary rat hippocampal neurons (b, f, g, h), MDCKcells (c, i) and mouse dendritic cells (d, e) and imaged with TIRFmicroscopy. e) Time sequence of a dendritic cell chemotaxing towards achemokine source. f) Cortical actin network of a hippocampal neuron. g)Time series of filopodial dynamics in a hippocampal neuron. h) Dynamicsnapshot of a filopodium: green shows signals that decrease in intensitybetween two frames (3 s apart), red shows signal that increases in thesame time span. j) Time series of an MDCK cell undergoing cytokinesis,showing Lifeact-G staining in the contractile ring. j) Time series ofactin patch movement within the cortex of a fibroblast. l-n) Functionalinfluence of transiently expressed Lifeact-GFP. Open bars:untransfected; grey bars: Lifeact-G; black bars: actin-GFP. Bars showvalues +/−SE (I) or +/−SD (m, n). (I) Neuronal polarization 3 days aftertransfection. m) Velocity of lamellipodial retrograde actin flow infibroblasts. n) Chemotactic speed of dendritic cells. Numbers are givenin percent relative to untransfected cells. o) Widefield microscopy ofMDCK cells double transfected with actin RFP and Lifeact-G. Scale bars:5 μm except 3j: 1 μm.

FIG. 4 F-Lifeact staining in fixed and living samples. MDCK cells (a)and cryo sections of mouse skeletal muscle (b) were fixed with PFA anddouble stained with F-Lifeact and phalloidin-Cy3. Mouse embryonicfibroblasts (c) and human primary neutrophil granulocytes (d) werescrape loaded with F-Lifeact, re-plated and subsequently visualized withTIRF microscopy. Scale bars: 5 μm.

FIG. 5 Actin dynamics in leukocytes

a, b) Time series of primary human neutrophils scrape loaded withF-Lifeact spreading on plated immune complexes. c, d) kymographs of theindicated regions in (a) and (b), respectively. e) Lifeact-G expressingmouse dendritic cell undergoing lamellipodial extension and retraction.f) Kymograph of the indicated region in e). Solid lines: retrogradeactin flow, dotted line: lamellipodial extension and dashed line:lamellipodial retraction. Size bars: 5 μm.

EXAMPLES 1. Material and Methods

Yeast strains and plasmid constructions. All Abp140-GFP plasmids wereexpressed in the S288C yeast background and based on the pRS315 backbone(LEU2, CEN). Expression was driven by the native Abp140 promoter (300bp) and a short GS linker was generated between Lifeact and GFP. Forexpression in mammalian cells the Lifeact sequence was cloned intopEGFP-N1 (Clonetech) or the respective plasmid with mRFPruby replacingGFP. In both cases the linker GDPPVAT was generated between Lifeact andthe fluorescent protein. Actin-GFP was used in the same plasmidbackbones. Cells were grown at 24° C. and observed from logarithmicallygrowing cultures (OD<0.8). Standard yeast media and procedures wereused.

Microscopy and image acquisition. TIRF images were captured on aniMic-stand from Till photonics with an 1.45 NA 100× objective fromOlympus. A 300 mW Argon laser and a 20 mW DPSS 561 nm laser wereselected through an AOTF. A 2-axis scan head (Yanus II) was used todigitally adjust the TIRF angle. Images were collected with a cooledImago QE CCD camera. Acquisition was controlled by the TILLvisIONsoftware package. Confocal images were collected on a standard Leica SP2setup. Epifluorescence images were collected on a Zeiss axiovert 200Mstand equipped with a climate control chamber from EMBL.

Image processing and data analysis. All image processing steps wereperformed in Metamorph (Molecular Devices). For visualization purposessequences were routinely processed by sequential application of a localbackground subtraction filter and a 3×3 gauss low pass filter. Coloroverlays and kymographs were created with the respective functions inMetamorph.

Proteins. Muscle actin, non-muscle actin, α-actinin and pyrene actinwere purchased from Cytoskeleton, Inc. Actin was diluted to workingconcentrations in G buffer (2 mM Tris HCl, pH 8.0, 0.2 mM ATP, 0.1 mMCaCl₂, 0.5 mM DTT).

Actin binding. Polymerization of actin was induced by addition of 0.1volume 10×KMEI buffer (50 mM KCl, 1 mM MgCl₂, 1 mM EGTA, 10 mM imidazoleHCl, pH 7.0) and incubation for >1 h at room temperature (RT). 44 μM ofF— Lifeact was incubated 30 min with F-actin and then spun 30 min at350,000×g at room temperature. The supernatant was removed and thepellet resuspended in 100 μl of 1×KMEI buffer. The amount of peptide wasmeasured in a Cary Eclipse Fluorescence Spectrophotometer withexcitation/emission set for FITC at 495 nm/520 nm. The bound/total ratiowas calculated as the signal from the pellet divided by the totalsignal. The K_(d) was obtained by fitting to a hyperbolic curve.

Binding to G-actin was determined from a spectral scan of pyrene actinin the presence of varying amounts of F-Lifeact. Averages of 5 emissionscans between 370 nm and 500 nm were used with excitation set to 365 nm.The bound/total ratio was calculated from the absolute emissiondifference at 385 nm between a given Lifeact concentration and thecontrol divided by the maximum difference observed. The K_(d) wasobtained by fitting to a hyperbolic curve.

Actin polymerization and depolymerization. For polymerization assays,20% pyrene-labelled actin was centrifuged at 350,000×g for 30 min at 24C to remove any nucleation seeds. Ca to Mg exchange was done adding 0.1Vof 10×ME buffer (50 μM MgCl₂, 0.2 mM EGTA) for 2 min. Polymerization waspromoted by addition of 0.1V 10×KMEI buffer. The final volume was 100μl. Pyrene fluorescence was monitored spectroscopically (excitation 365nm, emission 407 nm). To test the effect of F-Lifeact on polymerizationdifferent amount of F-Lifeact were added to the pyrene-actin aftercentrifugation and incubated for 5 min. Depolymerization was measured bymonitoring pyrene fluorescence after diluting 100% pyrene-labelledF-actin in 1×KMEI buffer to <0.2 μM. To test its effect ondepolymerization the indicated concentrations of F-Lifeact werepre-incubated with F-actin for 5 min before dilution.

Far UV CD Spectroscopy. CD measurements were performed on a Jasco J-715spectropolarimeter with a peptide concentration of 4.4 μM.

NMR sample preparation and spectroscopy. For NMR F-Lifeact was dissolvedin PBS pH 7.1. Unlabelled Lifeact was dissolved in PBS at pH 3. In orderto stabilize secondary structure of the peptide, 15% (v/v) of1,1,1,3,3,3-hexafluoro-2-propanol-d₂ (HFP-d₂) was added to the sample ofthe unlabeled peptide. 10% of D₂O (v/v) was added to all samples. NMRmeasurements were carried out at 600 MHz on a Bruker DRX-600spectrometer equipped with a cryoprobe at 300K. 2D nuclear Overhausereffect (NOESY) spectra were carried out with mixing time of 100 ms, andtotal correlated spectroscopy (TOCSY) spectra were recorded with DIPSI2mixing sequence of 35 ms and 80 ms duration (for the unlabelled peptidein alcohol/water and labelled peptide in PBS). Water suppression wascarried out using the WATERGATE sequence. Sequence specific resonanceassignments were carried out as in²¹. Amino acids spin systems wereidentified by analysis of TOCSY spectra. NOESY spectra were used toobserve contacts <5 Å.

Cell culture. Mouse primary dendritic cells were generated from flushedbone marrow suspension as described previously'. At day 8-10 cells werenucleofected using the primary mouse T cell kit and the Amaxanucleoporator according to the manufacturers recommendations andimmediately after transfection 200 ng/ml LPS were added over night andcells were subsequently sorted into GFP positive and negative fractionsby using fluorescent activated cell sorting. MDCK cells and mouseembryonic Fibroblasts were grown according to standard procedures andtransfected using lipofectamine.

Chemotaxis assays. PureCol™ gels containing mature dendritic cells werecast as described previously. Gels were overlayed with 50 μl of 0.6μg/ml CCL19 (R&D Systems) in RPMI, 10% FCS and imaged on an invertedAxiovert 40 (Zeiss) microscopes, equipped with custom built climatechambers (5% CO2, 37° C., humidified). Under agarose dendritic cellmigration was performed as described'. Briefly, dendritic cells wereadded into wells punched into an agarose layer. Recombinant CCL19 wasadded into the adjacent well and, following the chemokine gradient, thecells entered the space between agarose and cover slip. Chemotaxingcells were subsequently imaged with TIRF microscopy.

Culturing, transfection and classification of primary hippocampalneurons. Primary hippocampal neurons were isolated from rat embryos aspreviously described. Briefly, hippocampi derived from E18 rats weredissected, trypsinized, and dissociated. Directly after dissociation,5×10⁵ hippocampal neurons were transfected by the Amaxa nucleofectorsystem using 3 μg of highly purified plasmid DNA of pEGFP-Lifeact,pEGFP-actin or pEGFP-N2. The neurons were then immediately plated ontopoly-L-lysine-coated glass coverslips in 6 cm petri dishes containingminimal essential medium (MEM) and 10% heat-inactivated horse serum. Thecells were kept in 5% CO₂ at 36.5° C. 6-12 hrs after plating the glasscoverslips were then transferred into 6 cm petri dishes containingastrocytes in MEM and N2 supplements and kept in culture for 2 DIV.

Neurons were categorized into three stages: neurons with a single axon,multiple axons or no axon. A neurite length >35 μm was used as thresholdto define the axon. Developmental stages were observed by live cellmicroscopy as described.

Human Neutrophils and IC Induced Spreading

Human peripheral blood neutrophils were isolated by densitycentrifugation using a Pancoll™ gradient. Briefly, 10 ml bloodcontaining EDTA was diluted in 10 ml PBS and layered on 10 ml pancoll.After 30 min centrifugation at 500 g neutrophil were separated from theerythrocyte rich pellet by dextran sedimentation. Residual erythrocyteswere eliminated by hypertonic lysis and after washing in PBS,neutrophils were resuspended in RPMI containing 0.5% low endotoxinbovine serum albumin. Neutrophil purity was routinely ˜95% as assessedby forward and side scatter with flow cytometry as well as bymorphological analysis.

To form ICs in vitro, glass slides were coated with 5 mg/ml ovalbumin inPBS overnight at 4° C. followed by washing and incubation in rabbitanti-ovalbumin serum at 50 μg/ml specific IgG for 2 h at roomtemperature. F-Lifeact loaded neutrophils were subjected to ICs in thepresence of 10 ng/ml tumour necrosis factor-α to study actinreorganization in response to ICs.

2. Results

2.1 Identification of a novel F-actin binding domain in Abp140

Among all actin binding proteins from the yeast S. cerevisiae onlyAbp140-GFP has been shown consistently to localize to both actin patchesand actin cables (17,18). As Abp140 is expressed at low levels and actincables are mostly oriented along the cell cortex, it is very difficultto visualize Abp140-GFP labelled cables with conventionalepifluorescence microscopy and we decided to use Total InternalReflection (TIRF)-microscopy instead. With this technique we were ableto improve image contrast greatly and to observe the full extent ofcortical actin distribution for the first time (FIG. 1 a). The improvedsignal to noise ratio also allowed us to image actin dynamics at hightemporal resolution. Interestingly we found that at frame rates above10/s Abp140-GFP exhibited a strong blinking behaviour (FIG. 1 b)indicating a relatively low affinity of Abp140-GFP to actin.

Abp140 has a highly conserved methyltransferase domain at its C-terminusand a charged N-terminal half that has no apparent similarity toproteins form higher eukaryotes. Using TIRE imaging as readout we setout to identify the actin binding domain of Abp140 through serialdeletions. When we fused various domains of Abp140 to GFP we weresurprised to find that a short N-terminal peptide of only 17aa wassufficient to achieve actin localization comparable to the fullengthprotein (FIG. 1 c, d). An even shorter 11 aa peptide still retainedactin binding properties but was nearly exclusively restricted to actinpatches. This might be due to reduced actin affinity of the peptide sothat only structures of high actin density could be stained.Interestingly, in contrast to the rest of the N-terminal part of Abp140,the identified 17aa peptide is highly conserved in Abp140-homologuesfrom closely related fungi (FIG. 1 e), indicating that this shortsequence constitutes a conserved actin binding motif. No other proteinin yeast or any other organisms contains related sequences. Theidentified peptide is the shortest marker for actin in living cellsidentified so far. We termed it Lifeact and since it was so effective instaining F-actin structures in yeast cells we reasoned that Lifeactmight be a good candidate for a general actin marker in eukaryoticcells. To this end we constructed mammalian expression vectors forLifeact-GFP and Lifeact-mRFPruby and also chemically synthesized the17aa peptide and tagged it with fluorescein (F-Lifeact) at itsN-terminus.

2.2 Biochemical Properties of the Lifeact Peptide

An effective probe for actin should have no or very little influence onthe dynamics of actin polymerization and depolymerization. We thereforetested the properties of F-Lifeact in in vitro assays for actindynamics. To determine the affinity of Lifeact to filamentous actin weperformed an F-actin co-sedimentation assay. The amounts of peptide inthe pellet and in the supernatant were measured spectroscopically.F-Lifeact bound to muscle actin with a dissociation constant (K_(d)) of3.1±0.7 μM (mean±95% Cl, n=3, FIG. 2 a). The K_(d) for non-muscle actinwas 5.4±2.6 μM. This low affinity is consistent with the blinkingbehaviour observed in yeast cells. We also determined whether F-Lifeactbinds to G-actin by monitoring fluorescence quenching of pyrene-labelledactin as a function of peptide concentration. We found that F-Lifeactbound to G-actin with a similar affinity of 3.0±0.8 μM (FIG. 2 b).

We tested possible effects of the peptide on actin polymerization inpyrene assays (19). Concentrations from 1.1 μM to 11 μM of Lifeact hadno visible effect on nucleation and elongation of F-actin (FIG. 2 c).Only at 44 μM a difference in nucleation could be observed where the lagphase of polymerization was reduced from 100 s to approximately 30 s(FIG. 2 c, black circles). Even at 44 μM peptide the elongation rate wasnot affected. We also tested potential effects on actin depolymerizationby diluting pyrene labelled F-actin below the critical concentration inthe presence of peptide. The difference in depolymerization rates ofF-actin incubated with either low (1.1 μM) or high (44 μM)concentrations of F-Lifeact was less than 10% (FIG. 2 d).

To investigate whether Lifeact adopts a specific structure that allowsit to bind to actin we examined its secondary structure by Far-UVCircular Dichroism (CD) spectroscopy and NMR. F-Lifeact dissolved in PBShad a slight propensity of adopting an α-helical structure (FIG. 2 eblue line), which could be stabilized by increasing amounts oftrifluorethanol (TFE) (FIG. 2 e). Titration with TFE showed the typicalα-helix polarization pattern (local minimum at 222 nm) at only 10% (FIG.3 e) indicating a strong tendency of F-Lifeact to form an α-helix.Unlabelled Lifeact peptide was not soluble in PBS and was thereforedissolved in 10% acetic acid (pH 3). In this buffer Lifeact did not showany helical properties (FIG. 2 e inset). However, addition of 15%hexafluoro-2-propanol (HFP) stabilized the secondary structure ofLifeact and analysis of 2D NOESY NMR spectra of HFP stabilized peptideshowed a typical α-helix signature from residues 2 to 10 (FIG. 2 f redbars). In contrast, F-Lifeact at neutral pH formed a nascent helixcovering residues 1-7 (FIG. 2 f blue bars), where only short range NH—NHNOE contacts were observed (20). These features of Lifeact arereminiscent of the behaviour of thymosin β₄, a 42 amino acid G-actinbinding peptide, which forms a nascent helix in water that can befurther stabilized by the addition of an alcohol (21). Crystallographicanalysis shows that one of nascent helices of thymosin β₄ becomes afully stable α-helix after binding to G-actin (22,23).

2.3 Use of Lifeact-GFP in Mammalian Cells

To express Lifeact in mammalian cells we introduced the coding DNA forthe 17aa peptide into the pEGFP-N1 backbone. The resulting plasmidexpressing a Lifeact-GFP fusion (Lifeact-G) from the strong CMV promoterwas then transiently transfected in a wide range of primary andimmortalized mammalian cell types derived from different species. Wecovered all major cell lineages by transiently expressing Lifeact-G inimmortalized mouse embryonic fibroblasts, Madin-Darby canine kidney(MDCK) cells (immortalized epithelial cells), primary rat hippocampalneurons and primary mouse dendritic cells (haematopoietic lineage of themyeloid type).

In all cells tested we obtained a clear and contrasted signal on F-actinstructures that matched the patterns previously reported for these celltypes (FIG. 3 a-d). We noted no signs of cytotoxicity and transiently aswell as stably transfected lines of MDCK and mouse embryonic fibroblastsmaintained their normal morphology and did not show signs of growthretardation.

We next wanted to test if Lifeact-G is a universal marker of F actin orif it selectively associates with subsets of actin structures. TIRFmicroscopy was used to image cortical F-actin structures dynamicallywith high resolution. In fibroblasts and dendritic cells the flow ofdynamic lamellipodial actin was prominent at the cell periphery (FIG. 3a, e), while stable stress fibres formed in the cell body of tightlyadherent fibroblasts (FIG. 3 a). Neurons were rich in dynamic filopodia(FIG. 3 b) that frequently underwent kinking (FIG. 3 g) and torsion(FIG. 3 h). The surface of neuronal cell bodies was covered in anisotropic network of actin filaments (FIG. 3 f) that was also seen inall other cell types. MDCK cells showed stress fibres andcircumferential actin belts at the cell periphery (FIG. 3 c). Duringcytokinesis Lifeact-G highlighted the contractile rings of MDCK cellsobserved by conventional wide field optics (FIG. 3 i). In all four celltypes we also saw numerous bright F-actin patches that associated withthe cortical network of actin cables (FIG. 3 f) and were highly dynamic.In most cases the behaviour of theses dots was reminiscent of actinpatches in S. cerevisiae with patches appearing and disappearing withoutsignificant lateral motion, but occasionally we could observe dots thatmoved directionally over larger distances (FIG. 3 j). To test ifLifeact-G compromises cytoskeletal functions quantitatively, we measuredthree parameters that are sensitive readouts for actin dynamics:neuronal polarization, retrograde flow within the leading lamella offibroblasts and directed migration of dendritic cells along gradients ofchemokine. In all cases we compared Lifeact-G with actin GFP expressedfrom the same vector (CMV promoter). Neuronal polarization was slightlyaffected by the expression of Lifeact-G (FIG. 3 l, 60.1±0.2% cellsformed one axon compared to 69±9% of mock transfected cells), but muchless than by a comparable expression of actin-GFP (52±4%). The speed ofretrograde flow in lamellipodia of Lifeact-G transfected fibroblasts wasindistinguishable from untransfected cells at 4 μm/min, whereas it wasreduced to about half in actin-GFP expressing cells (FIG. 3 m). FinallyLifeact-G had no effect on the speed of chemotactic dendritic cellmigration in a 3-dimensional collagen matrix, while actin-GFP expressingcells migrated slightly slower and less directionally (FIG. 3 n and notshown).

Next we directly compared the quality of Lifeact-G labelling to that ofthe actin GFP fusion. We performed double transfections with Lifeact-Gand actin-mRFPruby (FIG. 3 o) (24) or with Lifeact-mRFPruby andactin-GFP (not shown) in fibroblasts. Both probes showed overlappingpatterns with slightly different characteristics: while in TIRFmicroscopy both labels gave a clear signal, the actin-GFP was slightlyblurred in wide field optics due to high background signal from G-actin.In contrast, Lifeact-G signal showed high contrast indicating thatG-actin is not significantly bound by the peptide in living cells.Consequently the retrograde flow of lamellipodial actin in fibroblastswas easily visible with Lifeact-G in a widefield setup, whereasactin-GFP could not be used to track actin flow reliably (not shown).

2.4 Labelling of Cells with Fluorescent Peptides

The short Lifeact peptide can be readily chemically synthesized in largeamounts. We used the fluorescein conjugated Lifeact (F-Lifeact) to labelcells independently of genetic approaches. First, we tested if F-Lifeactcould stain fixed cells and tissue sections. To compare F-Lifeact withthe commonly used F-actin probe phalloidin directly, we performed doublestaining with Cy3-conjugated phalloidin on paraformaldehyde (PFA) fixedMDCK cells. Using TIRF microscopy to visualize the cell surfaceselectively, we observed nearly complete overlap of the two markers onthe patch-like structures of the cortical actin network and in stressfibres (FIG. 4 a). We further double stained PFA fixed tissue sectionsof heart and skeletal muscle from mice and found a similar overlap ofthe actin probes in a banded pattern (FIG. 4 b). These findingsdemonstrate that the F-Lifeact peptide can be used as a non-toxicequivalent to phalloidin.

We next used the F-Lifeact peptide for loading into live cells andsubsequent dynamic imaging. We performed so-called “scrape loading”(ref) of fibroblasts, MDCK cells and human neutrophils. This procedureallows diffusion of the peptide into the cytoplasm through transientmembrane pores caused by mechanical removal of adherent cells from theculture dish. To avoid artefacts due to membrane damage after scrapeloading or excessive loading of cells with peptide we concentrated onweakly labelled cells and used TIRF microscopy to observe actinstructures. Fibroblasts scrape loaded with F-Lifeact revealed thetypical distribution of F-actin in stress fibres and lamellipodia (FIG.4 c). The fluorescence signal was maintained over 4-6 hours beforedegradation of peptide and/or fading of the fluorochrome. In scrapeloaded human neutrophils we were able to show for the first time therapid dynamics of actin in these cells that exhibited extensiveundirected lamellar protrusions after attaching to the culture dish(FIG. 4 d).

2.5 Biological Validation

Neutrophils represent key players of the innate immune response andcontribute to the tissue repair system of the body (25). The rapidpolymerization of actin filaments is fundamental to neutrophil effectorfunctions, e.g., extravasation, chemotaxis and phagocytosis. Sinceneutrophils are terminally differentiated and thereforenon-transfectable, the current knowledge about neutrophil actinreorganization in response to chemotactic stimuli is mainly based onstudies using HL-60 cells, a neutrophil-like myeloid tumour cell line(26). Furthermore, the integrin-dependent cytoskeletal reorganization inresponse to immune complex (IC) deposits has only been studied on fixedcells using fluorescent phalloidin reagents (27). Using F-Lifeact wewere able, for the first time, to show actin dynamics in isolated humanneutrophils and during their spreading on ICs.

To validate the new probe we analyzed F-Lifeact loaded human neutrophilsspreading on immune complexes. Using TIRF microscopy we observed two Factin populations in the spreading cells. Peripheral areas spread outrapidly with a speed of 14.1±2.8 μm/min (n=10), while there were nosigns of retrograde actin transport (FIG. 5 a, c). In central areasstationary patches formed that rapidly extended into the periphery aftercells stopped spreading (FIG. 5 b, d, extension stopped at the 10 s timepoint).

We next analyzed Lifeact-G transfected dendritic cells during phases oflamellipodial protrusion. Kymographic analysis in an area oflamellipodial protrusion and subsequent retraction (FIG. 5 e, f) showedthat dendritic cells do not show a clear distinction between a leadinglamellipodium and a lamella, in contrast to data recently published forfibroblasts. We could not detect signs of periodic extension-retractioncycles as was demonstrated in spreading fibroblasts. Force generationfor lamellipodial extension clearly did not depend on retrograde actinflow, as during extension the actin network did not move relative to thesubstrate. Hence, lamellipodial extension occurred with velocities (4μm/min) that were comparable with the speed of retrograde flow duringretraction or stagnation of the cell edge. Extension in the absence ofretrograde flow is in line with recent findings that leukocyte migrationdoes not depend on receptor mediated force coupling of the leading edgeto the substrate (28).

3. Discussion

We have developed a novel actin probe, Lifeact, which is superior tocurrently available probes in several ways. Lifeact only consists of17aa and represents the smallest available actin probe to date. It iseasy to synthesize, both as oligonucleotide for generation of fusionproteins or as chemically labelled peptide. This makes Lifeact derivedprobes cost effective and widely accessible. The simple synthesis of alinear 17aa peptide, which can then easily be derivatized opens up newpossibilities for the generation of actin-directed markers or drugs. Asa consequence of its small size Lifeact can be easily delivered intocells. Transgenic fusion of the Lifeact coding sequence to fluorescentproteins yields higher expression levels than large cDNAs. Wesuccessfully introduced Lifeact-G into all major mammalian celllineages. We also found that F-Lifeact peptide can be used to stainactin in fixed or live mammalian cells as well as in Xenopus laevisoocytes (our unpublished observation). Lifeact, therefore, is analternative to the widely used cyclic peptide actin-probe phalloidin,which is usually purified from its biological source, the mushroomAmanita phalloides, because its synthesis is difficult.

The signal we obtained from Lifeact-G was specific for F-actinstructures and exhibited very low cytosolic background. In contrastactin GFP was usually expressed at lower levels and signals sufferedfrom high background staining of G-actin, especially when usingwidefield optics. Likewise, using F-Lifeact peptide we did not observeany nonspecific labelling in histology as well as in both PFA fixed andliving cells.

The most important advantage of Lifeact as an actin probe in livingcells is the lack of detectable interference with cellular processes. Wecould not measure significant effects of Lifeact expression oncytoskeletal dynamics, cell polarization or cell migration when weexpressed Lifeact-G in living cells. Even sensitive processes such asneuronal polarization or leukocyte chemotaxis were undisturbed in thepresence of high levels of Lifeact while lower fluorescent levels ofactin GFP significantly influenced both processes. The observation thatLifeact does not alter actin dynamics at the cellular level isconsistent with its low binding affinity to actin in vitro and the lackof effects on actin polymerization and depolymerization. This feature isunique among available actin probes and makes it non toxic which is anadvantage during production and handling. In addition Lifeact has nohomologous sequences in higher eukaryotes, which makes competition withendogenous proteins less likely.

REFERENCES

-   1. Bamburg, J. R. & Wiggan, O. P. ADF/cofilin and actin dynamics in    disease. Trends Cell Biol 12, 598-605 (2002).-   2. Laing, N. G. Congenital myopathies. Curr Opin Neurol 20, 583-589    (2007).-   3. Suresh, S. Biomechanics and biophysics of cancer cells. Acta    Biomater 3, 413-438 (2007).-   4. Weinzierl, G. et al. Regulation of cell separation in the    dimorphic fungus Ustilago maydis. Add Microbiol 45, 219-231 (2002).-   5. Waterman-Storer, C. M., Desai, A., Bulinski, J. C. &    Salmon, E. D. Fluorescent speckle microscopy, a method to visualize    the dynamics of protein assemblies in living cells. Curr Biol 8,    1227-1230 (1998).-   6. Schmit, A. C. & Lambert, A. M. Microinjected fluorescent    phalloidin in vivo reveals the F-actin dynamics and assembly in    higher plant mitotic cells. Plant Cell 2, 129-138 (1990).-   7. Yamada, S., Pokutta, S., Drees, F., Weis, W. I. & Nelson, W. J.    Deconstructing the cadherin-catenin-actin complex. Cell 123, 889-901    (2005).-   8. Edwards, K. A., Demsky, M., Montague, R. A., Weymouth, N. &    Kiehart, D. P. GFP-moesin illuminates actin cytoskeleton dynamics in    living tissue and demonstrates cell shape changes during    morphogenesis in Drosophila. Dev Biol 191, 103-117 (1997).-   9. Bretschneider, T. et al. Dynamic actin patterns and Arp2/3    assembly at the substrate-attached surface of motile cells. Curr    Biol 14, 1-10 (2004).-   10. Lenart, P. et al. A contractile nuclear actin network drives    chromosome congression in oocytes. Nature 436, 812-818 (2005).-   11. Pang, K. M., Lee, E. & Knecht, D. A. Use of a fusion protein    between GFP and an actin-binding domain to visualize transient    filamentous-actin structures. Curr Biol 8, 405-408 (1998).-   12. Burkel, B. M., von Dassow, G. & Bement, W. M. Versatile    fluorescent probes for actin filaments based on the actin-binding    domain of utrophin. Cell Motil Cytoskeleton 64, 822-832 (2007).-   13. Kost, B., Spielhofer, P. & Chua, N. H. A GFP-mouse talin fusion    protein labels plant actin filaments in vivo and visualizes the    actin cytoskeleton in growing pollen tubes. Plant J 16, 393-401    (1998).-   14. Sheahan, M. B., Staiger, C. J., Rose, R. J. & McCurdy, D. W. A    green fluorescent protein fusion to actin-binding domain 2 of    Arabidopsis fimbrin highlights new features of a dynamic actin    cytoskeleton in live plant cells. Plant Physiol 136, 3968-3978    (2004).-   15. Holweg, C. L. Living markers for actin block myosin-dependent    motility of plant organelles and auxin. Cell Motil Cytoskeleton 64,    69-81 (2007).-   16. Ketelaar, T., Anthony, R. G. & Hussey, P. J. Green fluorescent    protein-mTalin causes defects in actin organization and cell    expansion in Arabidopsis and inhibits actin depolymerizing factor's    actin depolymerizing activity in vitro. Plant Physiol 136, 3990-3998    (2004).-   17. Asakura, T. et al. Isolation and characterization of a novel    actin filament-binding protein from Saccharomyces cerevisiae.    Oncogene 16, 121-130 (1998).-   18. Yang, H. C. & Pon, L. A. Actin cable dynamics in budding yeast.    Proc Natl Acad Sci U SA 99, 751-756 (2002).-   19. Cooper, J. A., Walker, S. B. & Pollard, T. D. Pyrene actin:    documentation of the validity of a sensitive assay for actin    polymerization. J Muscle Res Cell Motil 4, 253-262 (1983).-   20. Dyson, H. J., Rance, M., Houghten, R. A., Wright, P. E. &    Lerner, R. A. Folding of immunogenic peptide fragments of proteins    in water solution. H. The nascent helix. J Mol Biol 201, 201-217    (1988).-   21. Czisch, M., Schleicher, M., Horger, S., Voelter, W. &    Holak, T. A. Conformation of thymosin beta 4 in water determined by    NMR spectroscopy. Eur J Biochem 218, 335-344 (1993).-   22. Hertzog, M. et al. The beta-thymosin/WH2 domain; structural    basis for the switch from inhibition to promotion of actin assembly.    Cell 117, 611-623 (2004).-   23. Irobi, E. et al. Structural basis of actin sequestration by    thymosin-beta4: implications for WH2 proteins. EMBO J 23, 3599-3608    (2004).-   24. Fischer, M., Haase, I., Wiesner, S. & Muller-Taubenberger, A.    Visualizing cytoskeleton dynamics in mammalian cells using a    humanized variant of monomeric red fluorescent protein. FEBS Lett    580, 2495-2502 (2006).-   25. Nathan, C. Neutrophils and immunity: challenges and    opportunities. Nat Rev Immunol 6, 173-182 (2006).-   26. Weiner, O. D. et al. Spatial control of actin polymerization    during neutrophil chemotaxis. Nat Cell Biol 1, 75-81 (1999).-   27. Tang, T. et al. A role for Mac-1 (CDIIb/CD18) in immune    complex-stimulated neutrophil function in vivo: Mac-1 deficiency    abrogates sustained Fcgamma receptor-dependent neutrophil adhesion    and complement-dependent proteinuria in acute glomerulonephritis. J    Exp Med 186, 1853-1863 (1997).-   28. Smith, L. A., Aranda-Espinoza, H., Haun, J. B., Dembo, M. &    Hammer, D. A. Neutrophil traction stresses are concentrated in the    uropod during migration. Biophys J 92, L58-60 (2007).-   29. Janke, C. et al. A versatile toolbox for PCR-based tagging of    yeast genes: new fluorescent proteins, more markers and promoter    substitution cassettes. Yeast 21, 947-962 (2004).-   30. Wüthrich, K. NMR of Proteins and Nucleic Acids (John Willey, New    York 1986).-   31. De Hoop, M. J., Meyn, L. & Dotti, C. G. in Cell Biology: A    Laboratory Handbook. (ed. J. E. Cells) (Academic Press, San Diego,    Calif.; 1997).-   32. Bradke, F. & Dotti, C. G. in Microinjection and Transgenesis:    Strategies and Protocols. (ed. A. C.-A a. A. Garcia-Carranca) 81-94    (Springer-Verlag, Heidelberg; 1998).

1. A peptide having the amino acid sequence (I) (M)_(n)GVADLIKKFE

wherein n is 0 or 1, or a variant thereof.
 2. The peptide of claim 1,wherein amino acid residues 2-10 form an α-helical structure.
 3. Thepeptide of claim 1 or 2 which binds to actin.
 4. The peptide of claim 3,wherein the dissociation constant K_(d) is ≦50 μM, preferably ≦10 μM andmore preferably ≦5 μM.
 5. The peptide of any one of claims 1-4 havingthe amino acid sequence (II) (M)_(n)GVADLIKKFESISKEE

wherein n is 0 or 1, or a variant thereof.
 6. The peptide of any one ofclaims 1-5, which has an amino acid sequence derived from fungalspecies.
 7. The peptide of any one of claims 1-6, which has a length ofup to 120, preferably up to 50, and more preferably up to 20 amino acidresidues.
 8. The peptide of any one of claims 1-7, which is coupled toat least one heterologous molecule.
 9. The peptide of claim 8, whereinthe heterologous molecule is a heterologous peptide or polypeptide. 10.The peptide of claim 9, wherein the heterologous molecule is afluorescent polypeptide such as GFP or a variant thereof.
 11. Thepeptide of claim 8, wherein the heterologous molecule is a non-peptidicmolecule.
 12. The peptide of claim 11, wherein the heterologous moleculeis a fluorescent labelling group such as fluorescein or rhodamin. 13.The peptide of any one of claims 1-12 which is multimeric.
 14. Thepeptide of any one of claims 8-13, wherein the heterologous molecule iscoupled to the N- and/or C-terminus of the peptide.
 15. A nucleic acidmolecule encoding a peptide of any one of claim 1-7, 9, 10 or
 13. 16.The nucleic acid molecule of claim 15 which is operatively linked to anexpression control sequence.
 17. A recombinant cell transfected ortransformed with a nucleic acid molecule of claim 15 or
 16. 18. Anon-human transgenic organism transfected or transformed with a nucleicacid molecule of claim 15 or
 16. 19. Use of a peptide of any one ofclaims 1-14, a nucleic acid molecule of claim 15 or 16, a recombinantcell of claim 17 or a non-human transgenic organism of claim 18 for thedetection and/or modulation of actin.
 20. Use of a peptide of claim 19within a cell, particularly within a living cell.
 21. Use of claim 19 or20 for the detection and/or modulation of actin-associated processessuch as cell migration, cell division and/or cell differentiation. 22.Use of a peptide of any one of claims 1-14, a nucleic acid molecule ofclaim 15 or 16, a recombinant cell of claim 17 or a non-human transgenicorganism of claim 18 in drug screening.